Adenovirus belongs to the family Adenoviridae first isolated in 1953. Human adenoviruses are categorized into six (6) subgenera (A through F) based on the genome similarity, oncogenecity, and blood coagulation characteristics. Adenoviruses infect most non-divided cells such as muscle, lung, brain, and cardiac cells, and its molecular biological characteristics are well known in the art. Its genome is composed of a linear, double-stranded DNA of 35 kb and its replication in the host cell depends on viral protein, E1A.
Above characteristics of adenovirus can be exploited by using a nonreplicative vector having E1A deleted therefrom. Since the development of HEK293 cell line in which the adenoviral E1 gene is inserted, the adenovirus vector system has been used in numerous studies, which has led to the development of virotherapeutics that utilize cytotoxicity of the host cell. Oncorine®, which is the oncolytic virotherapeutics commercialized in China in 2005, is an E1B55-defective adenovirus for selectively inducing apoptotic cell death in p53-defective tumor.
In the development of selective replicative adenovirus therapeutic agents, selective expression of E1A protein is most important, and there have been suggested many cases regarding possible tumor-selective adenovirus gene therapeutic agents using tumor selective expression promoter. Most of the tumor-selective adenoviruses are prepared using the commonly-found human adenovirus serotype 5 (“HAd5”). It has been reported that human has high levels of adenovirus neutralizing antibodies, since HAd5 occupies 80% of prevalence (Appaiahgari, M. B., et al. (2007) Clinical and Vaccine Immunol. 14, 1053-1055). These neutralizing antibodies against adenoviral capsid protein influence the efficacy and toxicity of the adenovirus systemically administered (Chen, Y., et al. (2000) Hum. Gene Ther. 11, 1553-1567).
Viral capsid consists of three (3) kinds of proteins, i.e., hexon, fiber, and penton, and comprises a capsomere having a symmetric icosahedron structure consisting of 240 hexons and 12 pentons. Each penton binds a protruded trimeric fiber of 70˜100 nm. When infected by adenovirus, the trimeric fiber attaches to Coxackie Adenovirus Receptor (CAR) on the surface membrane of host cell in the process of adenovirus infection. The RGD region of penton binds to integrin, which leads to viral absorption and penetration into the host cell.
It has been reported that Loop 1 (L1) and Loop 2 (L2) of a hexon protein are exposed on the outside of the viral capsomere structure. L1 and L2 respectively contain six (6) hypervariable regions (HVRs), i.e., HVR-1 to HVR-6 within the 132nd to 320th amino acids and seventh HVR (HVR-7) within the 408th to 459th amino acids of the hexon protein.
The adenoviruses provide an elegant and efficient means of transferring therapeutic genes into cells. However, one problem encountered with the use of adenoviral vectors for gene transfer in vivo is the generation of antibodies to antigenic epitopes on adenoviral capsid proteins.
When adenovirus is administered to human body, neutralizing antibodies against hexon proteins are formed, and such antibodies mostly target the dominant HVR regions. It is also known that the antibodies reduce the efficiency of viral replication by way of inhibiting the infection of host cells (Wohlfart, C. (1988) J. Virol. 62, 2321-2328, Toogood, C.I.A., et al. (1992) J. Gen. Virol. 73, 1429-1435. Sumida, S. M., et al. (2005) J. Immunol. 174, 7179-7185).
The problems caused by the preponderance of human neutralizing antibodies against HAd5 in human must be overcome when administering adenovirus using a viral gene delivery vector and viral therapeutic agent. In addition to the above mentioned problems associated with the neutralizing antibodies, it has been reported that adenoviruses infect the liver when exposing systemically. In this connection, adenovirus has been reported to have hepatotropism, and when adenoviruses are administered via an intravenous route, 90% thereof is transferred to the liver within 24 hours (Worgall, S., et al. (1997) Hum. Gene Ther. 8, 37-44). Due to such hepatoselectivity of the adenovirus, in 1999, young patient, Jessie Gelsinger, under a clinical trial using gene therapeutic adenovirus agents succumbed due to acute hepatotoxicity. Thus, dose of adenovirus has been restricted to an amount that does not exceed 1×1013 vp since then. Therefore, hepatotoxicity is generally considered as a dose-limiting factor in nonclinical/clinical trials for many gene therapeutic agents using adenovirus (Alemany, R. et al. (2001) Gene Ther., 8(17), 1347-1353; Christ, M., et al. (2000) Hum. Gene Ther., 11(3), 415-427; Lieber, A., et al. (1997) J. Virol., 7(11), 8798-8807). Such liver selectivity is a major problem in achieving efficient cure by systemic administration of an adenoviral therapeutic agent (Worgall, S., et al. (1997) Hum. Gene Ther., 8, 37-44).
In this regard, Waddington et al. have recently reported that Gla domain, blood coagulating factor, combines with hexon protein of adenovirus in blood, which facilitates adenovirus transfer to the liver (Waddington, S. N., et al. (2008) Cell, 132, 397-409). It has been speculated that HVR-3, HVR-5 or HVR-7 of hexon can combine with blood coagulating factor, Gla domain (Kalyuzhniy, O., et al. (2008) Proc. Nat'l Acd. Sci. 105, 5483-5488). The HVR varies depending on the serotype of adenovirus, and it is not clear yet what is the crucial factor for binding affinity to blood coagulating factor. It has been reported that the maximum tolerated dose of adenovirus can be raised tenfold by way of inserting a specific protein such as RGD, RFP, and BAP (Biotin Acceptor Peptide) to significantly weaken binding affinity to blood coagulating factor and to reduce the hepatotropism (Shashkova, E. V., et al. (2009) Mol. Ther. 17, 2121-2130).
A number of functions of the hexon protein are now known, and many studies to modify hexon proteins in order to overcome the problems of hepatotoxicity and anti-adenoviral immunity are currently being conducted. There are four strategies for modifying hexon protein: 1) replacing the hexon gene with the corresponding hexon gene of other adenovirus serotype, 2) inserting a peptide into HVR, 3) replacing the gene encoding HVR of a hexon protein with the corresponding gene encoding HVR of other adenovirus serotype, and 4) removing the region from the HVR that binds the blood coagulating factor and neutralizing antibody. Up to date, the method of inserting a peptide into the HVR and the method of replacing the gene encoding HVR with the corresponding gene encoding HVR of other adenovirus serotype are carried out for hexon modification. Among above mentioned four strategies, complete hexon substitution is most apparent method to change viral immunogenicity. However, a method to achieve complete hexon exchange for modifying hexon protein has the problem of deteriorated productivity due to the fact that subtle structural differences in binding hexon to penton and fiber induce instability of the adenoviral capsid structure (Roberts, D. M., et al. (2006) Nature, 441, p239-243; Youil, R., et al. (2002) Hum. Gene. Ther. 13, p311-320; Shashkova, E., et al. (2009) Mol. Ther 17, 2121-2130).
Further, intense studies for the modification of capsid protein using serotypes of heterogenous adenovirus as well as those of human adenovirus are in progress. It has been reported that the prevalence rate of neutralizing antibodies against chimpanzee adenoviruses pan 5, 6, 7 and 9, classified as simian adenovirus serotypes 22 to 25, respectively, is less than 6%, and therefore, a simian adenoviral vector system including chimpanzee adenovirus can be useful as a gene therapeutic vector (Roy, S. et al. (2004) Hum. Gene Ther. 15, p519-530). International Patent Publication Nos. WO 2006/040330 and WO 2002/083902 teach the use of the fiber or hexon protein of human serotypes 11, 24, 26, 30, 34, 35, 48, 49, and 50 for suppressing immune response caused by neutralizing antibodies in the recombinant chimeric adenovirus where the adenoviral knob domain binding to the CAR or a hexon protein is substituted with those of other serotypes. Regarding simian adenovirus serotype, International Publication No. WO 2005/001103 discloses a chimeric adenovirus using simian adenovirus serotype 18.
However, there exists a strong need to develop an adenovirus having lower immunogenicity and lower toxicity. Thus, the present inventors have identified a novel SAd19 hexon gene isolated from baboon excrements and have found that it is highly capable of evading the neutralizing antibodies against HAd5 and it exhibits a low toxicity.